Nutritional stress is commonly encountered by pathogenic bacteria such as Borrelia burgdorferi during growth in their hosts. This stimulus triggers a bacterial global regulatory response, the stringent response, which is mediated by the alarmon (p)ppGpp and modulates expression of many genes. In the previous funding period, we found that the B. burgdorferi bmp chromosomal gene cluster encoding the paralogous membrane Bmp lipoproteins BmpD, BmpC, BmpA and BmpB was present in all B. burgdorferi sensu lato and that it formed two complex transcriptional units transcribed into variable monocistronic and polycistronic messages to generate different mRNA and gene product concentrations. Our preliminary evidence now indicates that B. burgdorferi has a transcriptionally active relA/spoT gene needed for generation of (p)ppGpp and the presence of (p)ppGpp is associated with modulation of expression of bmp cluster genes under different environmental conditions. The genetic, transcriptional, and biological characteristics of the bmp gene cluster in the context of the reduced and condensed genome of B. burgdorferi makes it an ideal model to study chromosomal gene regulation by trans regulators such as (p)ppGpp and sigma factors and by cis DNA sequences. The hypothesis underlying this project is that expression of bmp and other B. burgdorferi genes is modulated in trans by the stringent response both directly by (p)ppGpp and indirectly through sigma-54 and sigma-S and in cis by the DNA sequences of their 5 upstream promoter regions. The specific aims of this project are: 1) characterize regulation of bmp expression by the RelA/SpoT-mediated stringent response during growth of B. burgdorferi in BSKH media, in co-culture with tick cells, and in rat peritoneal culture champers and ticks; 2) ascertain the roles of sigma-S and sigma-54 in modulating bmp expression by the stringent response in B. burgdorferi in vitro and in vivo; and 3) identify putative regulatory proteins binding to 5 upstream DNA sequences of the rpsL, bmpD, bmpC, bmpA and sigma-S genes and their DNA binding sites. The proposed array of molecular, functional and genetic experiments will permit us to identify global factors, hierarchical regulatory networks and cis acting DNA sequences involved in the regulation of bmp genes, and will take studies of gene expression and modulation in B. burgdorferi from a descriptive paradigm to a paradigm framed by mechanistic and causal interpretations.